Although there are commercial assays for detecting RNA in blood, the RNA detected in such assays is usually present in extracellular forms, such as HIV or HCV particles in the blood. Detection of RNA or other target molecules from within red blood cells is more challenging. Reagents used in lysis may interfere with subsequent processing as many non-target molecules released by lysis, particularly nucleases or proteases, may degrade target molecules.
The intrinsic instability of RNA and presence of RNAses in whole blood makes isolation of RNA a difficult task. The use of high purity, intact RNA facilitates sensitive clinical diagnostic assays. Existing approaches typically involve several sequential steps: a step to disrupt the cells, a step to denature the proteins, another step for the stabilization and protection of RNA from RNAses, and then a step for isolation of the RNA
Tetradecyltrimethylammonium oxalate (TDTMAO) is commonly used for transport, storage and processing of blood (U.S. Pat. Nos. 6,602,718 and 6,617,170). This quaternary amine is contained, for example, in the PAXgene™ Blood RNA System (BD Biosciences) and works by penetrating the cell and stabilizing intracellular target RNA. The RNA can then be later purified and analyzed from the components of whole blood using standard techniques. Methods for lysing cells and inhibiting RNases using guanidinium salts are also known (Chomczynski et al. (1987) Anal. Biochem. 162, 156-159).
Human Babesiosis is an emerging infectious disease resulting from a tick-borne intraerythrocytic infection of red blood cells. Babesia microti, the most common cause for Babesiosis in the United States is wide epidemic in the Northeastern and upper Midwestern states. This species has been implicated in a majority of the transfusion-transmitted Babesiosis (TTB) cases in the United States and is currently the most reported transfusion-transmitted disease to the FDA. Between 2005 and 2010, 3.6% of transfusion-related deaths reported to the FDA were attributed to TTB. However, there is no FDA approved test for blood screening for Babesia to date despite the risk that this agent poses to the United States blood supply.